CN114277121B

CN114277121B - Application of gene SDHAF4 as drug target for improving insulin sensitivity and controlling obesity

Info

Publication number: CN114277121B
Application number: CN202111632642.XA
Authority: CN
Inventors: 刘健康; 冯智辉; 王雪强
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2021-12-28
Filing date: 2021-12-28
Publication date: 2023-07-07
Anticipated expiration: 2041-12-28
Also published as: CN114277121A

Abstract

The application of the gene SDHAF4 as a drug target for improving insulin sensitivity and controlling obesity, wherein the drug comprises polypeptide or siRNA form, and promotes the down regulation of the expression of the liver SDHAF4 protein or the reduction of the protein activity; inhibiting the expression or function of the liver SDHAF4 protein, thereby inhibiting the assembly and maturation of SDH complex; the medicine is the activity inhibition of the liver SDHAF4 as a target; to activate urea/arginine/nitric oxide pathway to increase collective insulin sensitivity and reduce obesity; therefore, the gene SDHAF4 can be used as a target point for preparing medicines for regulating insulin sensitivity and controlling obesity so as to prepare medicines for regulating insulin sensitivity and controlling obesity better and more.

Description

Application of gene SDHAF4 as drug target for improving insulin sensitivity and controlling obesity

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to application of a gene SDHAF4 as a drug target for improving insulin sensitivity and controlling obesity.

Background

With the development of economy and the improvement of the living standard of people, metabolic related diseases, especially obesity and diabetes, caused by overnutrition are increasingly one of serious diseases threatening human health and life, and the development of medicaments and means for treatment is always the focus of research in the field of life science.

Metabolic syndrome is a pathological state of the organism characterized by obesity, hyperglycemia, hypertension, hyperlipidemia, etc., is an important disease foundation of heart and brain vascular lesions and diabetes, and has become a serious population health problem facing the China society. Glycolipid metabolic disorders are the main manifestation of metabolic syndrome, and involve imbalance of metabolism of various organs such as liver, skeletal muscle, fat, etc., in vivo, and in the development process, the mechanisms of induction and metabolic coordination of different tissues on glycolipid molecules are still unclear. Liver is taken as a main metabolism organ of the organism, and participates in important tasks such as digestion, oxidation removal, energy storage, secretory protein synthesis and the like in the organism, and mitochondria which are rich in the liver are key organelles for completing glycolipid metabolism by liver cells. Under physiological conditions, the process by which mitochondria maintain a dynamic balance of production, degradation, fusion, division, distribution, etc., and maintain normal function is called mitochondrial homeostasis (mitochondrial homeostasis). The maintenance of mitochondrial homeostasis can efficiently provide cells with the required energy, while intermediates produced during mitochondrial metabolism also become metabolic substrates and signaling molecules for cells to achieve complex vital activities. The liver perceives the content of glycolipid in vivo, and through intracytoplasmic treatment, synthesis or catabolism of glycolipid is finally realized by mitochondrial redox reaction, and at the same time, other metabolic organs in the body such as fat, skeletal muscle and the like can also perceive signals from liver metabolites, so that the metabolic processes of the liver can be coordinated. Liver mitochondria are also considered to be new targets for drug development of metabolic diseases.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the gene SDHAF4 as a drug target for improving insulin sensitivity and controlling obesity so as to prepare more drugs for regulating insulin sensitivity and controlling obesity.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

use of the gene SDHAF4 as a drug target for improving insulin sensitivity and controlling obesity, said gene SDHAF4 having a gene ID of 135154.

The medicine contains compounds, polypeptides and siRNA forms, and can promote down-regulation of liver SDHAF4 protein expression or reduction of protein activity.

The medicine inhibits the assembly and maturation of SDH complex by inhibiting the expression or function of liver SDHAF4 protein.

The drug is the activity inhibition of the liver SDHAF4 as a target spot, is characterized by inhibiting or preventing the combination of the SDHAF4 and SDHA/SDHB, and can inhibit the function and the activity of SDH.

The medicine promotes down-regulation of liver SDHAF4 expression to activate urea/arginine/nitric oxide pathway to raise collective insulin sensitivity and reduce obesity.

Compared with the prior art, the invention has the following beneficial technical effects:

experiments prove that the reduction of the expression level of the liver SDHAF4 can improve the insulin sensitivity of the organism, and the SDHAF4 is disclosed as a key molecule for the assembly and combination of SDHA and SDHB; and the reduction of the expression of the liver SDHAF4 leads to the reduction of the SDH activity, activates urea/arginine/nitric oxide metabolic pathway to compensate and supplement the SDH metabolic substrate fumaric acid, activates the nitric oxide pathway, increases the nitric oxide content in the systemic circulation, acts on the main metabolic tissues of the liver, muscle and fat of the organism, and improves the metabolic sensitivity of the organism. The inhibitor can reduce the metabolism sensitivity of the organism by inhibiting the generation of nitric oxide in the liver, and fully shows how the SDHAF4 gene influences the SDH activity so as to activate urea/arginine/nitric oxide metabolic pathways for compensation, thereby generating the key factor of regulating and controlling the metabolism sensitivity of the organism, namely nitric oxide. Therefore, the gene SDHAF4 can be used as a target point for preparing medicines for regulating insulin sensitivity and controlling obesity so as to prepare medicines for regulating insulin sensitivity and controlling obesity better and more.

Drawings

A in fig. 1 is a graph of the results of Real-timeRNA detection of mitochondrial respiratory complex key subunit mRNA levels (Gapdh as an internal reference, n=6, < p < 0.001); b is a western blot analysis result plot of liver tissue (GAPDH as internal reference, n=6, < p < 0.001); c is a liver tissue immunoprecipitation detection result graph; d is a graph of the results of liver mitochondria detection of complex activity on mitochondrial respiratory chain (n=6, ×p < 0.001).

A in fig. 2 is a graph of fasting blood glucose test results; b is a graph of glucose tolerance detection results of 3 and 8 month old liver knockout SDHAF4 mice (n=8, ×p < 0.01); c is a graph of the results of the detection of pyruvic acid tolerance in 3 and 8 month old liver knockout SDHAF4 mice (n=8, ×p < 0.01); d is a graph of the results of the 3 and 8 month old liver knockout SDHAF4 mice insulin resistance assay (n=8, ×p < 0.01); e is a graph of the analysis result of insulin signal paths in the liver, muscle and fat of the mice.

A in fig. 3 is a graph showing the results of weight gain curves of mice induced by a 16-week high fat diet; b is a graph of body weight and tissue weight analysis results; c is brown fat, inguinal fat, epididymal fat and perirenal fat HE staining; d, glucose tolerance detection result graphs; e is an insulin resistance assay result graph (n=8, < p < 0.01); f is a graph of results of biochemical detection of insulin sensitivity in liver, muscle and fat (n=3, p < 0.01).

A in fig. 4 is a graph of liver metabonomics KEGG analysis results; b is a graph of the analysis result of the metabolic substrate in the arginine-urea-NO metabolic network; c is an arginine pathway gene mRNA level detection result graph; d is an arginine pathway gene protein level detection result diagram; e is a graph of the glucose tolerance test results of mice after L-NAME treatment.

Detailed Description

The invention will now be described in further detail with reference to specific examples, which are intended to illustrate, but not to limit, the invention.

The experimental means and experimental procedures required for the present invention are provided below.

1. Western immunoblotting and immunoprecipitation

The total protein was extracted by lysing the mouse tissue, protein separation was performed on SDS-PAGE gels, and stained onto nitrocellulose membranes. After blocking 1 hour at room temperature with 1% bovine serum albumin, incubation with the specific primary antibody was carried out overnight at 4 ℃. The following day, after incubation with horseradish peroxidase (HRP) -crosslinked secondary antibodies for 1 hour at room temperature, the bands were developed on a chemiluminescent instrument (BioRad, hercules, CA, USA).

From within mouse tissue lysates

Among the antibodies used were phospho-AKT (4060), AKT (4691), GAPDH (5174), SDH5 (45849), SDHA (5839) all available from CellSignaling Technology (Beverley, mass., USA); antibody NDUFA9 (PA 5-83598), UQCRC1 (459140), COX4 (MA 5-15078), ATP5A (459240) are all available from LifeTechnologies (USA); antibody SDHB (178423), SDHAF4 (122196), SDHC (155999) and sdhd (189945) are all purchased from Abcam (Cambridge, UK); antibodies SDHA (390381) and sdhb (271548) are both purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA); secondary antibodies Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (115-035-003), peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-003) were all purchased from Jackson (USA).

2. Real-time quantitative PCR

Total RNA was extracted from mouse tissue using TriPure Isolation Reagent (Roche, basel, switzerland) and then reverse transcribed into cDNA using the kit (BioRad, hercules, calif., USA). Real-time quantitative PCR reactions were performed using iQSYBR Green Supermix (BioRad) and data analysis was performed using CFX Connect real-time PCR detection system (BioRad).

3. Adenovirus vector construction and packaging

Wild type SDHAF4 cDNA was cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific) to construct the entry vector. Then the entry vector and pAD/CMV/V5-DEST vector are subjected to recombination reaction to construct the adenovirus vector. Adenovirus packaging and amplification were performed in 293A cells. Mice were then given tail vein injections.

4. Animal experiment

Liver-specific knockout mice were generated using the Cre/loxP system. Sdhaf4f/f mice were prepared by Beijing Bai Aosai map Co., ltd. Using conventional homologous recombination in embryonic stem cells, two sgRNAs were designed to create a chromosome deletion of 3kb (exons 1-2) at the Sdhaf4 locus of the mouse genome. The F1 generation mutant is identified by PCR and southern blot method, and allele cloning and sequencing are carried out. All mice were backcrossed with C57BL/6 mice for at least 7 passages. Alb-Cre mice were obtained from Jackson lab (003574). Both male and female mice exhibited similar physiological phenotypes, and only data for male mice were reported. Mice were genotyped using a custom TaqMan genotyping assay (GeneStar). The tail DNA was extracted with proteinase K lysis buffer (100 mm Tris-HCl (pH 8.0), 5 mm EDTA (pH 8.0), 200 mm sodium chloride, 0.2% SDS,0.1 mg/ml proteinase K). The DNA was then analyzed by PCR.

For adenovirus-mediated expression of Sdhaf4 in mouse liver experiments, 8 week old liver-specific knockout mice were used. Adenovirus was packaged with 293A cells and purified by CsCl ultracentrifugation. The control adenoviruses used were prepared simultaneously. After virus titer determination, tail vein injection (5×10 per mouse ⁸ Individual viral plaque forming units (pfu)). The test was performed 2 weeks after injection, and tissues and plasma were collected for analysis.

5. Statistical analysis

Data are expressed as mean ± SEM. The data were analyzed using Prism (GraphPad). Two pairs of the two-tailed student T tests are adopted for comparison. Other data correct for multiple comparisons using one-or two-factor analysis of variance. In all cases, p less than 0.05 is considered significant.

The relevant results of the experiments of the present invention are given below.

Control of metabolic homeostasis is essential for maintaining the physiological function and health of the body. Oxidative phosphorylation of semi-autonomous organelle mitochondria by coupling of electron transport chains (OXPHOS) to form proton electrochemical gradients across the inner mitochondrial membrane provides about 95% of the ATP required for the cell, a key role played in the change in metabolic rhythms. Mitochondrial dysfunction is associated with a variety of human metabolic diseases including fatty liver disease, diabetes, neurodegenerative disease, cardiovascular disease, and the like. Therefore, it is important to improve the understanding of the proteins in mitochondria of living beings. We found that when the initial metabolic rhythm in the mouse liver is remodeled, the assembly and activity of the SDH complex is reduced. SDH is a highly conserved heterotetramer that is involved in both electron transport by the mitochondrial electron transport chain and in the tricarboxylic acid cycle. It catalyzes the oxidation of succinic acid to fumaric acid in the tricarboxylic acid cycle, reduces ubiquitin to ubiquitin alcohol in the oxidative phosphorylation electron transfer chain, pumps out protons to establish a mitochondrial membrane potential difference for ATP synthesis. SDH anchored heterodimers SDHC and SDHD intercalate into the Inner Mitochondrial Membrane (IMM) with SDHA and SDHB as catalytic subunits facing the mitochondrial matrix. The membrane anchor domain comprising five redox cofactors, one iron sulfur (FeS) cluster of covalently bound FAD and three hydrophilic segments and one heme-containing membrane anchor domain is essential for its catalytic function. Four assembly factors: SDHAF1, SDHAF2, SDHAF3, and SDHAF4.SDHAF1 mutations are associated with infant leukoencephalopathy. SDHAF2 has been shown to be necessary for covalent binding of FAD to the catalytic SDHA subunit. The loss of function mutation of the human SDHAF2 gene is associated with hereditary paraganglioma disease. Mutations in SDHAF3 may be associated with increased prevalence of pheochromocytomas or paragangliomas. Yeast lacking SDHAF3 and drosophila have impaired SDH activity, while SDH2 (SDHB in humans) levels are reduced. However, SDHAF4 has recently been reported as a cofactor, specifically interacting with the catalytic Sdh1 (SDHA in humans) subunit in the mitochondrial matrix, facilitating its association with Sdh2, and subsequently assembling Sdh full complexes in yeast, drosophila and mammalian cells. SDHAF4 is critical to prevent drosophila motor defects and neurodegeneration. However, the changes in mammals that lead to SDHAF4 mutations are not yet known. In this patent it was found that SDHAF4 is essential for SDH to occur in eukaryotic organisms. Knocking out SDHAF4 in liver can enhance metabolism sensitivity of adult mice, and can reduce obesity caused by high-fat diet.

SDHAF4 is a key factor in SDH assembly and is critical to mitochondrial activity

To verify the function of SDHAF4 in mammals, liver-specific knockout mice were generated using the Cre/loxP system. After the SDHAF4 is specifically knocked out by the liver, the protein stability of each subunit of SDH in the liver is obviously affected, the deletion of the SDHAF4 obviously reduces the formation of SDHA and SDHB dimers, the expression level of other subunits is obviously reduced, and the activity of a compound II is also obviously reduced (figure 1). Four graphs in fig. 1 illustrate that SDHAF4 is a key factor in SDH assembly, critical to mitochondrial activity; extracting RNA after liver cleavage of an 8-week-old liver-knocked-out SDHAF4 mouse to detect the mRNA level of SDHAF4 and key subunits of each mitochondrial respiratory complex, wherein the result shows that the mRNA level of the complex subunits on the mitochondrial respiratory chain in the liver of the liver-knocked-out SDHAF4 mouse is not significantly changed (Gapdh is taken as an internal reference, n=6, p < 0.001); b, extracting total protein after liver cleavage of an 8-week-old liver knockout SDHAF4 mouse for Westernblot analysis, and showing that the liver knockout SDHAF4 results in reduced expression level of mitochondrial respiration complex II Subunit (SDH) (GAPDH is used as an internal reference, n=6, p < 0.001); c, extracting total protein for immunoprecipitation detection after liver cleavage of the mouse with the liver knockdown SDHAF4 of 8 weeks old, and the result shows that the mouse liver knockdown SDHAF4 reduces assembly combination of SDHA and SDHB; d, detecting the activity of the complex on the mitochondrial respiratory chain by extracting liver mitochondria of the SDHAF4 mouse with the age of 8 weeks, and the result shows that the activity of the complex II is obviously reduced, and meanwhile, the complexes III, IV and V are also affected by different degrees (n=6, p < 0.001).

Liver knockout SDHAF4 mice have enhanced metabolic sensitivity

Liver is used as an important metabolic organ, in order to explore which influences the activity change of key enzymes in mitochondria have on organisms, the weight, ingestion, glucose tolerance, insulin tolerance and pyruvic acid tolerance of mice after the liver-specific knockout of SDHAF4 are detected, and the result shows that the liver-specific knockout of SDHAF4 has lighter weight and higher metabolic sensitivity than the control mice. The mice were stimulated with insulin and the primary metabolic tissues including liver, fat and muscle were examined for p-AKT levels (fig. 3), respectively, and the results showed that liver-specific knockout SDHAF4 mice had significantly elevated p-AKT levels of liver, fat and muscle. The above results indicate that the deletion or inhibition of liver SDHAF4 can have a good effect of improving metabolic sensitivity of the mouse body (fig. 2).

Five graphs in fig. 2 illustrate the enhancement of metabolic sensitivity in liver knockout SDHAF4 mice. a, after the liver of the mouse knocks out SDHAF4, the fasting blood glucose is normal; b, glucose tolerance assays of 3 and 8 month old liver knockout SDHAF4 mice showed a significant enhancement in their metabolic capacity (n=8, ×p < 0.01); c, liver knockout SDHAF4 mice at 3 months of age and 8 months of age showed a significant enhancement in their metabolic capacity (n=8, ×p < 0.01); d,3 and 8 month old liver knockout SDHAF4 mice showed significant enhancement of insulin sensitivity (n=8, ×p < 0.01); the ingestion gap of the mice is not obvious; e, after the SDHAF4 mice with 3 months of age are injected with insulin for 15 minutes, insulin signal paths in livers, muscles and fat are detected, and the insulin signal activation in tissues is obviously higher than that of normal mice.

The mouse liver can resist obesity caused by high-fat diet after knocking out SDHAF4

To further verify the status of SDHAF4 knockout mice against metabolic stress, the knockout mice and control mice given a high fat diet simultaneously, and the results showed that under the high fat diet condition, the liver knockout SDHAF4 mice had lighter weight than the control group, and were mainly represented by accumulation of fat globules, and glucose tolerance and insulin tolerance of the liver knockout SDHAF4 mice were also stronger than those of the control group, and similarly, p-AKT levels of liver, muscle and fat of the liver knockout SDHAF4 mice were higher in biochemical test (fig. 3). The above experiments demonstrate that liver knockout SDHAF4 is resistant to obesity and metabolic disorders caused by high fat diets.

Six graphs in fig. 3 demonstrate that mice were resistant to high fat diet induced obesity and insulin resistance after liver knockout of SDHAF4. Feeding wild type mice with the age of 8 weeks and SDHAF4 type mice with the liver knocked out with a high-fat diet, and monitoring the weight and the physiological index of the mice; the results show that: (a) the weight of the liver of the mice after being knocked out of SDHAF4 is significantly different from that of the wild type control mice after being subjected to a high fat diet for 16 weeks, the weight of the mice in the knocked out group is lighter, anatomical detection results show that the weight difference of the two groups is mainly reflected in the weight of fat (b), pathological section detection is carried out on brown fat, inguinal fat, epididymal fat and perirenal fat of the mice fed with the high fat diet for 16 weeks, the result suggests that the mice in the knocked out group can reduce fat droplet accumulation (c) and reduce the total weight of fat after being knocked out of SDHAF4 by feeding with the high fat diet, meanwhile, the glucose tolerance (d) and insulin tolerance (e) of the knockout group are enhanced (n=8, ×p < 0.01) relative to the control group, and the biochemical detection results also show that the insulin sensitive glue control group of the liver, muscle and fat of the mice in the knockout group is increased (f) (n=3, ×p < 0.01).

Mouse liver knockout SDHAF4 activates arginine-urea-NO metabolic network

To further explain why the absence of SDHAF4 in the liver can cause a good metabolic phenotype in the body, analysis of the liver by metabonomics has found that the arginine-urea-NO metabolic network in the liver is significantly activated, which can effectively supplement the metabolite fumaric acid of SDH to maintain the stability of the liver mitochondrial tricarboxylic acid cycle. At the same time, the activation of the metabolic network effectively increases the release of NO in the liver, which can act on muscle, fat and other tissues to increase the insulin sensitivity, and finally promotes the increase of the metabolic capacity of the whole organism (fig. 4). The above experiments indicate that the absence of SDHAF4 from liver SDHAF4 can improve the interpretation of the mechanisms underlying metabolic capacity of the body,

five graphs in fig. 4 illustrate that mice liver knockdown activates the arginine-urea-NO metabolic pathway after SDHAF4. Liver metabonomics analysis showed a significant increase in the arginine metabolic pathway in the liver of the knockout SDHAF4 mice (a in fig. 4) and an increase in the metabolic substrate content in the arginine-urea-NO metabolic network (b in fig. 4). While both the gene mRNA level (c in fig. 4) and protein content of the relevant pathway were significantly elevated in knockout mice (d in fig. 4). Inhibiting synthesis of NO in liver can effectively reduce metabolic sensitivity of knockout mice (e in fig. 4), suggesting that activation of arginine-urea-NO metabolic network is key to improvement of insulin sensitivity and glucose metabolism ability of liver SDHAF4 mice.

Based on the above detection results, the present invention proposes the following applications:

the gene SDHAF4 is used as a target spot in the preparation of the medicine for improving insulin sensitivity and obesity.

Further: the medicine is used for promoting the down regulation of SDHAF4 expression in liver to inhibit the activity of SDH.

Alternatively, the drug is targeted to inhibit SDHAF4 activity, thereby inhibiting SDH 4 catalytic assembly SDH activity.

The gene SDHAF4 provided by the invention can be used as a target for preparing the drugs for improving insulin sensitivity and obesity and improving metabolic diseases.

The embodiments given above are preferred examples for realizing the present invention, and the present invention is not limited to the above-described embodiments. Any immaterial additions and substitutions made by those skilled in the art according to the technical features of the technical scheme of the invention are all within the protection scope of the invention.

Claims

1. An application of gene SDHAF4 as target in screening the medicines for improving insulin sensitivity and controlling obesity.

2. The use of claim 1, wherein the medicament comprises a polypeptide or siRNA form that promotes down-regulation of liver SDHAF4 protein expression or reduced protein activity.

3. The use according to claim 2, wherein the medicament inhibits the assembly and maturation of the SDH complex by inhibiting the expression or function of the liver SDHAF4 protein.

4. The use of claim 3, wherein the agent inhibits liver SDHAF 4-targeted activity, inhibits or prevents SDHAF4 binding to SDHA/SDHB.

5. The use of claim 3, wherein the agent promotes down-regulation of liver SDHAF4 expression to activate urea/arginine/nitric oxide pathway to increase collective insulin sensitivity and reduce obesity.